Induction And Genetic Analysis Of Mitotic Gynogenesis In Large Yellow Croaker Pseudosciaena Crocea

To establish the protocol for induction mitotic gynogenesis and provide basic geneticimformation of mitogens in large yellow croaker Pseudosciaena crocea, the conditions ofinduction of mitotic gynogenesis were optimized, and the genetic analyses of the families ofmitotic gynogenesis, meiotic gynogenesis and control group were also conduct withmicrosatellite and AFLP marks. The main results were shown as follows:1. The influence of temperature on the time of1st cleavage （τI） and intervals of eggcleavage （τ0） were determined. The research indicated that the higher the temperature, the1stcleavage was sooner, about3.1min sooner every1℃increased between19℃and28℃. Therelations between Ï„I and temperature was fit as Ï„0=432.83e^-T/8.19+22.06（R²=0.988, P<0.05）using the first-order exponential decay regression equation. Ï„0was shorter when the temperatureimproved. The relations between Ï„0and temperature was fit as Ï„0=1304.22e^-T/4.50+9.73（R²=0.999, P<0.05）. The ratio of Ï„I/Ï„o ranged between2.24-2.91, increased with temperature rising,but the increased amplitude were decreased with temperature increasing.2. The optimal conditions to induce mitotic gynogenesis by hydrostatic pressure shock ofthe eggs were determined in this part. Combining the hatching with its haploid control groups,the production of normal gynogenesis diploids were evaluated as an index of appropriate UVradiation dosage. The results suggested that the best exposure time was90s with the intensity of2057μw·cm-2·s-1, for the sperm genetic inactivation. The condition for suppressing the1stcleavage of eggs by hydrostatic pressure shock were optimize with one-factor and orthogonalexperiment. The results showed that the best starting time window of pressure shock wasbetween Ï„I-6min and Ï„I-4min post insemination, with the pressure intensity of40-44Mpa for aduration of3min.3. The genetic analyses of mitotic gynogenesis families were identified with microsatellitemarks. Two mitotic gynogenesis families Mitotic-F1and Mitotic-F2were produced by blockingthe1st cleavage of genetically developed eggs with hydrostatic pressure, and verified withmicrosatellite markers. The inheritance and segregation of10microsatellite loci in putativegynogenetic doubled haploids （GDH） in these two families were investigated. In Mitotic-F1,twenty genotypes have been observed in30assayed progenies with8microsatellite loci. And allof progenies of Mitotic-F1were demonstrated as GDH for exclusive maternal inheritance andhomozygous at each locus, suggesting the production of GDH was100%. In30tested offsprings in Mitotic-F2,27were demonstrated as GDH,2contained male parent specific band, and1remained undefined. Twelve genotypes were observed in the27GDHs in Mitotic-F2with4tested loci. In addition, the segregation of microsatellite markers in GDHs was consistent withthe expected ratio according to Mendel’s law of inheritance at all the loci except LYC0026andLYC0053. The research also indicated that the segregation mode of GDH were completelyidentical between LYC0002and LYC0014.4. The transmission and separation of Microsatellite and AFLP markers in mito-gynogeneticfamily （Mitotic-F）, meio-gynogenetic family （Meiotic-F） and normal control family （Control-F）were studied comparetively. No male-specific genes were detected in both Mitotic-F andMeiotic-F, indicating that gynogens were produced in100%and all individuals are true gynogenin these two families. In mitotic-F, all68assayed fries were homozygous for the7microsatelliteloci, proving that homozygote can be obtained in one generation by induction mito-gynogenesis.Average genetic similarity coefficient among individuals in mitotic-F was much lower than thosein Meiotic-F and Control-F, and genetic diversity between Mitotic-F and female parent was largethan that between Meiotic-F and female parent, which reflected that the heterozygous genes offemale parent segrated completely in Mitotic-F. In Meiotic-F, high level of heterogeneity wasobserved. All examined loci, excepting LYC0022, retained heterozyosity. The averageheterozygosity was caculated as0.714, similar to that in Control-F. Moreover, Eighteen out of30segregted loci were deviated from the expected ratio, while most of loci fit the Mendel ratio inMitotic-F and Control-F. The results suggested that the loci were exchanged betweenhomologous chromosomes at high proportion and heterozygous female parent genes remainednon-separation at high proportion. Genetic similarity indexes between Meiotic-F and femaleparet and among the inviduals of Meiotic-F both ranked first among three kinds of families. Inaddition, two AFLP loci, E-AAG/M-CAG₉and E-AAC/M-CAG₂,) were found closely linkedwith lethal gene.